Enzyme amplification assay

ABSTRACT

NOVEL BIOLOGICAL ASSAY METHOD FOR DETERMINING THE PRESENCE OF A SPECIFIC ORGANIC MATERIAL BY EMPLOYING A MODI-SPECIFIC FOR ONE OR A GROUP OF MATERIALS (HEREINAFTER REFERRED AS TO &#34;LIGANDS&#34;) AND BINDING AN ENZYME TO THE LIGAND OR LIGANF. D C UNTERFEIT TO PROVIDE AN &#34;ENZYME-BOUNDLIGAND OR LIGANG COUNTERFEIT TO PROVIDE AN &#34;ENZYME-BOUNDLIGAND,&#34; AN EXTREMELY SENSITIVE METHOD IS PROVIDED FOR ASSAYING FOR LIGANDS. THE RECEPTOR WHEN BOUND TO THE ENZYME-BOUND-LIGAND SUBSTANTIALLY INHIBITS ENZYMATIC ACTIVITY, PROVIDING FOR DIFFERENT CATALYTIC EFFICIENCIES OF ENZYME-BOUND-LIGAND AND ENZYME-BOUND-LIGAND COMBINED WITH RECEPTOR. THE RECEPTOR, LIGAND AND ENZYME-BOUND-LIGAND ARE COMBINED IN AN ARBITRARY ORDER AND THE EFFECT OF THE PRESENCE OF LIGAND ON ENZYMATIC ACTIVITY DETERMINED. VARIOUS PROTOCOLS MAY BE USED FOR ASSAYING FOR ENNZYMATIC ACTIVITY AND RELATING TO THE RESULT TO THE AMOUNT OF LIGAND PRESENT.

"United States Patent 3,817,837 ENZYME AMPLIFICATION ASSAY Kenneth E. Rubenstein, Palo Alto, and Edwin F. Ullman, Atherton, Califi, assignors to Syva Corporation, Palo Alto, Calif.

No Drawing. Continuation-impart of abandoned application Ser. No. 143,609, May 14, 1971. This application Nov. 6, 1972, Ser. No. 304,156

Int. Cl. Gtlln 31/14 US. Cl. 195-103.5 R 91 Claims ABSTRACT OF THE DISCLOSURE Novel biological assay method for determining the presence of a specific organic material by employing a modified enzyme for amplification. By employing receptors specific for one or a group of materials (hereinafter referred to as ligands) and binding an enzyme to the ligand or ligand counterfeit to provide an enzyme-bound- ]igand, an extremely sensitive method is provided for assaying for ligands. The receptor when bound to the enzyme-bound-ligand substantially inhibits enzymatic activity, providing for different catalytic efiiciencies of enzyme-bound-ligand and enzyme-bound-ligand combined with receptor.

The receptor, ligand and enzyme-bound-ligand are combined in an arbitrary order and the effect of the presence of ligand on enzymatic activity determined. Various protocols may be used for assaying for enzymatic activity and relating the result to the amount of ligand present.

CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of application Ser. No. 143,609, filed May 14, 1971, now abandoned.

BACKGROUND OF THE INVENTION Field of the Invention There is a continually pressing need for rapid, accurate qualitative and quantitative determinations of biologically active substances at extremely low concentrations. The purpose of the determination can be extremely varied. Today, there is a Wide need for determining the presence of drugs or narcotics in body fluids, such as saliva, blood or urine. In addition, in medical diagnosis, it is frequently important to know the presence of various substances which are synthesized naturally by the body or ingested. These include hormones, both steroidal and polypeptides, prostaglandins, toxins, as well as other materials which may be involved in body functions. Frequently, one is concerned with extremely small amounts and occassionally, with very small differences in concentrations.

To meet these needs, a number of ways have been de vised for analyzing for trace amounts of materials. A common method is to use thin layer chromatography (TLC). By determining the flow factors and using specific reagents, the presence of certain materials can be detected; in many instances, the particular material can be isolated and identified quantitatively, for example, by mass spectroscopy or gas phase chromatography. However, thin layer chromatography has a number of deficiencies in being slow, requiring a high degree of proficiency in its being carried out, being subject to a wide range of interfering materials, and suffering from sever fluctuations in reliability. Therefore, the absence of satisfactory alternatives has resulted in intensive research efforts to determine improved methods of separation and identification.

An alternative to thin layer chromatography has been radioimmunoassay. Here, antibodies are employed for specific haptens or antigens. A radioactive ana og employing a radioactive atom of high flux is used ant. bound ice to the antigen. By mixing an antibody with solutions of the hapten or antigen and the radioactive hapten or antigen analog, the radioactive analog will be prevented from binding to the antibody in an amount directly related to the concentration of the hapten or antigen in the solution. By then separating the free radioactive analog from the antibody boimd radioactive analog and determining the radioactivity of the separate components, one can determine the amount of hapten or antigen in the original solution.

The use of radioactive materials is not desirable for a variety of reasons. First, radioactivity creates handling problems and undesirable hazards. Secondly, the preparation of such compounds involves similar hazards, greatly enhanced by the much larger amounts of radioactive materials which are present. Because of their instability, the radioactive materials have only a short life. In addition, the use of radioactive materials requires a license from the Atomic Energy Commission, subjecting the licensee to review by the Commission as to the maintenance of minimum operating standards. These standards may change from time to time, so as to involve added expense and inconvenience to the licensee. Finally, the separation of the bound and unbound radioactive analog is difiicult ad subject to error. See, for example, Abraham, Prelim. Comm, 29, 866 (1969).

Besides the aformentioned materials, assays at extremely low concentrations would be desirable for a variety of pesticides, such as insecticides, bactericides, fungicides, etc., as well as other organic pollutants, both in the air and water. Organic pollutants may be assayed whenever a receptor can be devised and the pollutant is inert to the reagents employed.

Description of the Prior Art Use of radioimmunoassay is described in two articles by Murphy, J. Clin. Endocr. 27, 973 (1967); ibid., 28, 343 (1968). The use of peroxidase as a marker in an immunochemical determination of antigens and antibodies is found in Stanislawski et al., C. R. Acad. Sci. Ser. D. 1970, 271 (16), 1442-5. (CA. 74 1144 B). See also, Nakane, et al., I. of Histochem. and Cytochem. 14, 929 (1967) and Avrameas, Int. Rev. of Cytology, 27, 349 (1970). A general description of thin layer chromatography for assay may be found in Stahl, Thin Layer Chromatography, Springer Verlag, New York, 1969. See also, Peron, et al., Immunologic Methods in Steroid Determination, Appleton, Century Crofts, New York, 1970.

Also of interest are publications by Van Weemen, et al., FEBS Letters 14, 232 (1971), and Engvall, et al., Immunochemistry, 8, 871 (1971) concerned with immunoassays employing enzymes. See also US. Pat. No. 3,654,090. See also, Cinader, Proceedings of the Second Meeting of the Foundation of European Biochemical Societies, Pergamon, Oxford, 1967, vol. 11, chapter four.

SUMMARY OF THE INVENTION Detection of ligands is obtained at extremely low concentrations by using specific receptor sites for the ligand and enzyme amplification of ligand displacement. By bonding a ligand or a ligand counterfeit to an enzyme while retaining enzymatic activity and then combining the enzyme-bound-ligand to a receptor for the ligand, the presence and amount of ligand in an unknown solution may be readily determined. By competition for receptor sites between the enzyme-bound-ligand and the free ligand, the two ligand moieties being added to the receptor simultaneously or sequentially, the difference in enzymatic activity resulting from the presence or absence of ligand may be determined in accordance with a particular analytical scheme. This difference will be related to the amount of ligand present in the unknown solution. Enzymatic activity is easily determined in known ways by following the change in concentration of an enzyme substrate or product of the substrate by standard techniques.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS This invention provides a method for detecting or assaying extremely low concentrations of a wide range of organic materials by relating the presence of a particular unknown to enzymatic activity. An amplification is obtained by having a large number of molecules formed or transformed as a result of the presence of one molecule. This amplification is achieved by bonding the compound to be assayed or a counterfeit of the compound to an enzyme. This assemblage is referred to as an enzymebound-ligand. The particular molecule to be assayed is referred to as a ligand. Te ligand analog will include either a ligand which is modified by replacing a proton with a linking group to bond to the enzyme or a ligand counterfeit which is a ligand modified by other than simple replacement of a proton to provide a linking site to the enzyme. The ligand and the enzyme-bound-ligaud are both capable of binding in a competitive fashion to specific receptor sites. It should also be noted that other compounds of very similar structure may serve as ligands capable of competing for these sites, e.g., morphine glucuronide and codeine will compete with enzyme-boundmorphine for binding to certain types of morphine antibodies. In most instances, this is advantageous in permitting one to assay for a class of physiologically closely related compounds.

Various methods or protocols may be employed in assaying for a wide variety of ligands. Normally, the ligand, enzyme-bound-ligand and receptor will be soluble in the medium employed. The substrate(s) for the enzyme may or may not be soluble in the medium. In some situa tions it may be desirable to provide a synthetic substrate which is not soluble or employ an insoluble natural substrate.

In carrying out the assay, the enzyme-bound-ligand is combined with a high molecular weight receptor which results in inhibition of enzymatic activity. When a ligand and enzyme-bound-ligand are introduced into a solution containing ligand receptor, the enzymatic activity of the solution after the three substances are combined will be affected by the concentration of the ligand present in the solution. That is, the enzyme-bound-ligand and the ligand will compete for the receptor sites. The number of enzymebound-ligand molecules not inhibited by the receptor will be directly related to the number of ligand molecules present in the solution. One can achieve this in two Ways: 1) either by competition, whereby the enzyme-bound-ligand and ligand are introduced to the receptor substantially simultaneously; or (2) the enzyme-bound-ligand or ligand may be first added to the receptor, and the system allowed to come to equilibrium, and then the ligand added or enzyme-bound-ligand added respectively, in effect, to displace the material originally added from the receptor. Since the enzymatic activity will be diminished or inhibited when the enzyme-bound-ligand is bound to the receptor, the enzymatic activity of the solution will be directly related to the amount of ligand present in the solution.

The assay can be carried out, either by considering the effect of ligand on the rate at which enzyme-bound-ligand binds to receptor or the effect of ligand on the equilibrium between the reagents: enzyme-bound-ligand and receptor. Where enzyme-bound-ligand and ligand are present with receptor, one need not wait until equilibrium is achieved between the three species. If one measures the enzymatic activity at a specific time or interval of time from the time of combination of the three species, the enzymatic activity of the assay mixture will be a function of the effect of the ligand on the rate of binding of the enzyme-bound-ligand to the receptor. By determining standards under the same conditions, including the same time interval, employing different concentrations of ligand, a smooth standard curve is obtained.

By measuring the effect of the ligand on rate of binding, rather than the effect on equilibrium, a shorter time interval between the time of combining the reagents and unknown suspected of containing the ligand and the time for the determination will be involved, as compared with waiting until equilibrium is achieved. It is frequently found that reproducible values can be obtained in from 0.1 to 5 minutes after combining the reagents and unknown. The rate of enzymatic activity is usually determined over a short time interval, e.g., one minute. The time interval can be the second, third, etc. minute from the time when the reagents and unknown were combined.

The concentrations of the reagents: the enzyme-boundligand and the receptor, may be varied widely. Normally, the concentration of receptor (based on active sites) and enzyme-bound-ligand will be from about l0- to 10- M, more usually from 10- to 10- M. The lower limit for the concentration of enzyme-bound-ligand is predicated on the minimum amount which can be detected. This will vary with different enzymes as well as different detection systems.

The amount of receptor employed is normally calculated based on receptor sites and will vary with the concentration of enzyme-bound-ligand, the ratio of ligand to enzyme in the enzyme-bound-ligand, and the afiinity of the receptor for the ligand. Usually, there will be at least 1 active receptor site per molecule of enzyme-bound-ligand and less than about 20 active sites per molecule of ligand as enzyme-bound-ligand, but site-ligand molecule ratios may be as high as 1,000 to 1, depending on the type of assay and the affinity of the receptor. Preferably, the ratio of receptor active sites to molecules of enzyme-boundligand will be at least one, usually at least two, and the ratio of active sites to molecules of ligand as enzymebound-ligand will be less than about 5 to 1. The ratio will vary to a great degree depending on binding constants and the amount of ligand suspected of being present. The method of determining binding sites for the receptor will be discussed subsequently in the experimental section.

The enzyme-bound-ligand will usually have molecules of ligand to enzyme subunit ratios on the average over the entire composition in the range of 0101-1001, frequently 0.02-50: l, and more frequently about 0.04-25 1, wherein the number of ligands when the ligand is a protein is expressed as the number of ligand molecules times the number of its component polypeptide chains. For small ligands (less than about 10,000 molecular weight( there will generally be at least one ligand, more usually at least two ligands per enzyme, while with large ligands (greater than about 5,000 molecular weight) there will generally be at least one enzyme per ligand. In the area of overlap, the ratio will depend on the nature of the ligand, among other factors to be discussed.

The number of small ligands per enzyme will be affected to some degree by the molecular Weight of the enzyme. However, normally, the fewer molecules of ligand bound to an enzyme to achieve the desired degree of inhibitability with receptor, the more sensitive the assay. Therefore, the number of small ligands per enzyme will usually not exceed 40, more usually not exceed 30, and will not exceed l ligand per 2,000 molecular weight of enzyme on the average over the entire composition. Usually, the range of ligands will be 1 to 40, more usually 1 to 24, and with random substitution 2 to 20.

With large ligands, there will be on the average not more than one enzyme per 2,000 molecular weight, usually not more than one enzyme per 4,000 molecular weight, and more usually not more than one enezyme per 6,000 molecular weight.

In some instances, a number of enzymes bind together in a stable arrangement to form a multienzyme complex. Because of the juxtaposition of the enzymes, a number of reactions may be carried out sequentially in an efficient manner, providing localized high concentrations of reactants. Therefore, the ligand may be bound to a combination of enzymes, whereby there will be a plurality of enzymes per ligand. If a number of ligands were bound to the multienzyme complex, one could have 1:1 mole ratio of enzymes to ligand, although, in fact, there would be a plurality of enezymes and ligands involved in a single aggregation. The number of enzymes bound together, either as a multienzyme complex or by another mechanism will rarely exceed 20, usually not exceed 10, and commonly be in the range of 2 to 5 enzymes.

All other things being equal, the greater the number of enzymes per large ligand, the greater the sensitivity of the assay. However, the enzymes may interfere with receptor recognition, affect solubility and be deleterious in other ways. Therefore, usually, the number of enzymes bonded to a large ligand will be such that there will be no more than one enzyme polypeptide chain for every 2,000 molecular weight of the ligand.

The concentration of receptor and enzyme will be related to the range of concentration of the ligand to be assayed. The solution to be assayed will be used directly, unless a relatively high concentration of ligand is present. If a high concentration is present, the unknown solution will be diluted so as to provide a convenient concentration. However, in many biological systems of interest, the amount of material being assayed will be relatively small and dilution of the unknown substrate will usually not be required.

To illustrate the subject method, a soluble receptor is employed for a particular ligand. For illustrative purposes, the ligand will be considered the hapten, morphine, and the receptor will be an antibody specific for morphine. It should be noted parenthetically, that antibodies generally recognize molecular shaped and distribution of polyar groups in a ligand, although a portion of the ligand may be significantly modified without preventing recognition. For example, both morphine and its glucuronide can be bound to certain morphine antibodies.

An enzyme is first modified by bonding one or more morphine molecules to the enzyme; a sufficient number of morphine groups are employed so that greater than about 20% inhibition, usually 50% inhibition, and preferably, at least 70% inhibition is obtained when the maximum number of ligands are conjugated to receptor. Complete inhibition is usually neither necessary or desirable. In many instances, all that is required is that there be a measurable difference between completely uninhibited and maximally inhibited enzyme-bound-ligand which would allow for a semi-quantitative or quantitative determination of a ligand through a desired range of concentrations. Any convenient enzyme can be used that will catalyze the reaction of a substrate that can be easily detected and for which a substrate is available which allows for inhibition of the enzyme when bound to receptor.

A solution is prepared of the antibody of the requisite concentration. Only a few microliters of solution are required. The antibody, maintained at a pH at whichit is active in binding morphine, is introduced into a solution of the enzyme-bound-morphine at the desired concentration. The reactivity of the combined antibody and enzymebound-morphone solution can be determined by taking an aliquot, adding it to its substrate under conditions where the enzyme is active, and determining the spectroscopic change as a function of time at a constant temperature. The rate of this change will be the result that should be obtained when there is no morphine present in the unknown solution.

Normally, the ligand and enzyme-bound-ligand reversably bind to receptor, so that the order of addition of reagents is not crucial.

A second aliquot is taken and added to the unknown solution. The unknown solution may contain the substrate and any other additives which are required for enzymatic activity. Alternatively, the unknown solution may first be combined with the antibody-(enzyme-boundmorphine) complex, allowed to come to equilibrium and then mixed with the substrate. In either case the rate of change in the spectrum is determined. A variant of the above method is to add combined enzyme-bound-morphine and unknown solution to the antibody and then add this solution to the substrate. Other obvious variations come readily to mind.

If all concentrations of reagents except morphine are kept constant and several standard solutions of morphine are employed, then one can relate the change in the spectrum over specified periods of time to the morphine concentrations. Obviously, the standardized system can then be used to determine rapidly, accurately, and efliciently the amount of morphine, or any other ligand in the unknown.

The manner of assaying for the enzyme can be widely varied depending on the enzyme, and to some degree the ligand and the medium in which the ligand is obtained. Conveniently, spectrophotometric measurements can be employed, where absorption of a cofactor, a substrate or the product of the substrate absorbs light in the ultraviolet or visible region. However, in many instances other methods of determination may be preferred. Such methods include fluorimetry, measuring luminescence, ion specific electrodes, viscometry, electron spin resonance spectrometry, and metering pH, to name a few of the more popular methods.

The assays will normally be carried out at moderate temperatures, usually in the range of from 10 to C., and more usually in the range of about 15 to 40 C. The pH of the assay solutions will be in the range of about 5 to 10, usually about 6 to 9. Illustrative buffers include (tn'shydroxymethyl)methylamine salt, carbonate, borate and phosphate.

Whether oxygen is present or the assay is carried out in an inert atmosphere, will depend on the particular as say. Where oxygen may be an interferant, an inert atmosphere will normally be employed. Normally, hydroxylic media will be employed, particularly aqueous media, since these are the media in which the enzyme is active. However 0 to 40 volume percent of other liquids may also be present as co-solvents, such as alcohols, esters, ketones, amides, etc. The particular choice of the cosolvent will depend on the other reagents present in the medium, the effect on enzyme activity, and any desirable or undesirable interactions with the substrate or products.

As already indicated, antibodies will frequently recognize a family of compounds, where the geometry and spatial distribution of polar groups are similar. Frequently, by devising the haptenic structure and the method of binding to the antigen when producing the antibodies, the specificity of the antibody can be varied. In some instances, it may be desirable to use two or more antibodies, usually not more than six antibodies, so that the antibody reagent solution will be able to detect an entire group of compounds, e.g., morphine and barbiturates. This can beparticularly valuable for screening a sample to determine the presence of any member of a group of compounds or determining whether a particular class of compounds is present, e.g., drugs of abuse or sex hormones. When combinations of antibodies are used, it will usually be necessary to employ corresponding combinations of enzyme-bound-ligands.

Ligand Turning now to a general consideration of the reagents, the first reagent to be considered is the ligand. Any ligand may be employed for which an appropriate receptor may be found having satisfactory specificity for the ligand. The recent literature contains an increasing number of reports of receptors for an increasingly wide variety of biologically active materials. Compounds for which receptors can be provided range from simple phenylalkylamines, e.g., amphetamine, to very high molecular weight polymers, e.g., proteins.

Among ligands which are drugs, will be compounds which act as narcotics, hypnotics, sedatives, analgesics, antipyretics, anaesthetics, psychotogenic drugs, muscle relaxants, nervous system stimulants, anticholinesterase agents, parasympathomimetic agents, sympathomimetic agents, a-adrenergic blocking agents, antiadrenergic agents, ganglionic stimulating and blocking agents, neuromuscular agents, histamines, antihistamines, S-hydroxytryptamine and antagonists, cardiovascular drugs, antiarrhythmic drugs, antihypertensive agents, vasodilator drugs, diuretics, pesticides (fungicides, antihelminthics, insecticides, ectoparasiticides, etc.), antimalarial drugs, antibiotics, antimetabolites, hormones, vitamins, sugars, thyroid and antithyroid drugs, corticosteroids, insulin, oral hypoglemic drugs, tumor cells, bacterial and viral proteins, toxins, blood proteins, and their metabolites.

(A drug is any chemical agent that atfects living protoplasm. (Goodman & Gilman, The Pharmacological Basis of Therapeutics, 3rd ed., Macmillan, New York (1965).) A narcotic is any agent that produces sleep as well as analgesia.)

Included among such drugs and agents are alkaloids, steroids, polypeptides and proteins, prostaglandins, catecholamines, xanthines, arylalkylamines, heterocyclics, e.g., thiazines, piperazines, indoles, and thiazoles, amino acids, etc.

Other ligands of interest besides drugs are industrial pollutants, flavoring agents, food additives, e.g., preservatives, and food contaminants.

Broadly, the ligands will be organic compounds of from 100 to 100,000 molecular weight, usually of from about 125 to 40,000 molecular weight, more usually 125 to 20,000 molecular weight. The ligand will usually have from about 8 to 5,000 carbon atoms and from about 1 to 3,500 heteroatoms.

A substantial portion of the ligands will be monomers or low order polymers, which will have molecular weights in the range of about 100 to 2,000, more usually 125 to 1,000. Another significant portion of the ligands will be polymers (compounds having a recurring group) which will have molecular Weights in the range of from. about 750 to 100,000, usually from about 2,000 to 60,000, more usually 2,000 to 50,000. For polymers of varying molecular weight, weight average molecular weight is intended.

In some instances, high molecular weight materials will be of interest. For example, blood proteins will generally be in excess of 100,000 molecular weight. In the case of lipoproteins, the molecular weight will be in the range of 3 million to 20 million. The globulins, albumins and fibrinogens will be in the range of 100,000 to 1,000,000.

The ligands will normally be composed of carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorous, halogen, and metals, primarily as their cations, such as the alkali and alkaline earth metals and the metals of Groups IB, IIB, VIIB, and VIIIB, particularly the third row of the periodic chart. Most usually, the ligands will be composed primarily of carbon, hydrogen, nitrogen, oxygen and sulfur.

Structurally, the ligands may be monomers or polymers, acyclic, mono or polycyclic, having carbocyclic or heterocyclic rings. The ligands will have a wide variety of functionalities, such as halo, oxocarbonyl, nonoxocarbonyl, amino, oxy (hydroxy, aryloxy, alyloxy and cycloallyloxy [alyl intends a monovalent aliphatic radical]), thiooxy, dithio, hydrazo, and combinations thereof.

The ligands may be divided into three diiferent categories, based on their biological relationship to the receptor. The first category is antigens, which when introduced into the bloodstream of a vertebrate, result in the formation of antibodies. The second category is haptens, which when bound to an antigenic carrier, and'the hapten bound antigenic carrier is introduced into the bloodstream of a vertebrate, elicit formation of antibodies specific for the hapten. The third category of ligands includes those which have naturally occurring receptors in a living organism and the receptors can be isolated in a form specific for the ligand.

Of course, biological substances which are native to one species and have naturally occurring receptors in that species, may also be haptens when bonded to a protein and introduced into an animal of the same or a different species. Therefore, the classification is somewhat arbitrary in that the ligand may be an antigen as to one species, a hapten as to another species, and may have naturally occurring receptors in a third species.

Antigens are for the most part protein or polysaccharide in nature and foreign to the animal'into which they are injected.

The most important body of ligands for the purposes of the invention are the haptens. Substances which on injection do not give rise to antibodies, but which are able to react with antibodies specifically to produce either precipitation or to inhibit precipitation have been termed haptens. This definition has been used to include not only the simple chemical substances which are determinants of specificity when conjugated to protein, and which inhibit precipitation, but also substances obtained from natural sources such as the pneumococcal type specific polysaccharides and dextran which are not antigenic in the rabbit on primary injection. Kabat, et al., Experimental Immunochemistry, Charles C. Thomas, Springfield, Ill. (1967). In the following discussion the term hapten will be confined to groups artificially introduced into antigenic carriers which promote the formation of antibodies to those groups.

The third group of ligands are those which have naturally occurring receptors. The receptors may be proteins, nucleic acids, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or membranes associated with cells. Illustrative ligands which have naturally occurring receptors are thyroxine, many steroids, such as the estrogens, cortisone, corticosterone, and estradiol; polypeptides such as insulin and angiotensin, as well as other naturally occurring biologically active compounds. See Murphy, et al., J. Clin. Endocr., 24, 187 (1964), Murphy, ibid., 27, 973 (1957); ibid, 28, 343 (1969); BBA, 176, 262 (1969); McEwen, et al., Nature, 226, 263 (1970); Morgan, et al. Diabetes (1966); Page, et al., J. Clin. Endocr., 28, 200 (1969).

The ligands may also be categorized by the chemical families which have become accepted in the literature. In some cases, included in the family for the purpose of this invention, will be those physiomimetic substances which are similar in structure to a part of the naturally occurring structure and either mimic or inhibit the physio logical properties of the natural substances. Also, groups of synthetic substances will be included, such as the barbiturates and amphetamines. In addition, any of these compounds may be modified for linking to the enzyme at a site that may cause all biological activity to be destroyed. Other structural modifications may be made for the ease of synthesis or control of the characteristics of the antibody. These modified compounds are referred to as ligand counterfeits.

A general category of ligands of particular interest are drugs and chemically altered compounds, as'well as the metabolites of such compounds. The interest in assaying for drugs varies widely, from determining whether individuals have been taking a specific illicit drug," or have such drug in their possession, to determining what drug has been administered or the concentration of the drug in a specific biological fluid.

The drugs are normally of from eight carbon atoms to 40 carbon atoms, usually of from 9 to 26 carbon atoms, and from 1 to 25, usually from 1 to 10 heteroatoms, usually oxygen, nitrogen or sulfur. A large category of drugs have from one to two nitrogen atoms.

One class of drugs has the following basic functionality:

where the lines intend a bond to a carbon atom and wherein any of the carbon atoms and the nitrogen atom may be bonded to hydrogen, carbon or a heterofunctionality. Drugs which have this basic structure include the opiates such as morphine and heroin, meperidine, and methadone.

Another class of drugs are the epinephrine like drugs which have the following basic functionality:

where the lines intend a bond to a carbon atom and wherein any of the carbon atoms and the nitrogen atom may be bonded to hydrogen, carbon or a heterofunctionality. Drugs which have this basis structure include amphetamine, narceine, epinephrine, ephedrine and L-dopa.

The ligand analogs of drugs will usually have molecular weights in the range of 150 to 1,200 more usually in the range of 175 to 700.

Alkaloids The first category is the alkaloids. Included in the category of alkaloids, for the purpose of this invention, are those compounds which are synthetically prepared to physiologically simulate the naturally occurring alkaloids. All of the naturally occurring alkaloids have an amine nitrogen as a heteroannular member. The synthetic al- -kaloids will normally have a tertiary amine, which may or may not be a heteroannular member. The alkaloids have a variety of functionalities present on the molecule,

The opiates are morphine alkaloids. All of these molecules have the following functionality and minimum structures:

wherein the free valences are satisfied by a wide variety of groups, primarily carbon and hydrogen.

The enzyme-bound-ligand analog of these compounds will for the most part have the following minimum skel-- etal structure:

wherein X is a bond or a functionality such as imino, azo, oxy, thio, sulfonyl, oxocarbonyl, nonoxocarbonyl, or combinations thereof. Oxygen will be in the ortho, meta or 3 position. A is an enzyme which is bonded to X at other than its reactive site and retains a substantial portion of its natural enzymatic activity. There will be m ligands bonded through X to the enzyme A.

The enzyme-bound-morphine and its closely related analogs will have the following formula:

won

W -NW wa wa n wherein:

any one of the W groups can be X* or an H of any of the W groups may be replaced by X*, wherein X* is a bond or a linking group;

A* is an enzyme bonded at other than its reactive site, having a number (n) of ligands in the range of 1 to the molecular weight of A* divided by 2,000, usually in the range of 2 to 40;

W is hydrogen or hydrocarbon of from one to eight carbon atoms, particularly alkyl or alkenyl of from 1 to 4 carbon atoms, cycloalkylalkyl of from 4 to 6 carbon atoms, or aralkyl, e.g., methyl, allyl, 3-methylbut- 2-enyl-l, cyclopropylmethyl and fl-phenethyl;

W is hydrogen;

W is hydrogen;

W is hydrogen or taken together with W a divalent radical of from 3 to 6 carbon atoms and 0 to 2 oxygen atoms, forming a six membered carbocyclic ring with the carbon chain to which they are attached, e.g., proplylene 1,3,1-hydroxyprop-2-enylene-'1,3-hydroxypropylene 1,3, 1 acetoxypropylene 1,3, -l-acetoxyprop-2-enylene-1,3, 1-oxopropylene-1,3, 1-oxoprop-2- enylene1,3;

W is hydrogen or hydroxyl;

W is hydrogen, hydroxyl or taken together with W W is hydrogen or methyl;

W is hydrogen, methyl or hydroxyl;

W is hydrogen, hydroxy, acyloxy of from 1 to 3 carbon atoms, e.g., acetoxy, (unless otherwise indicated, acyl intends only nonoxocarbonyl), hydrocarbyloxy of from 1 to 3 carbon atoms, e.g., methoxy, ethoxy, 2-(N- morpholino)ethoxy and glucuronyl; and

W is hydrogen. (It is understood that in all the formulas, except when a minimum or skeletal structure is indicated, unsatisfied valences are satisfied by hydrogen.)

(Hydrocarbyl is an organic radical composed solely of hydrogen and carbon and may be saturated or unsaturated, aliphatic, alicyclic, aromatic or combinations thereof.)

By other than its reactive site, it is intended that the ligand is not bonded to the enzyme at a position which prevents the enzyme substrate, including necessary cofactors, from entering into the reaction catalyzed by the enzyme. It is understood, that with random substitution, the resulting product may include enzyme which has been 1 1 deactivated by ligand bonded at the reactive site, as well as enzyme which is active and has ligand bonded at other than the reactive site.

The close morphine analogs will have the following formula:

A N-w -1 site of ethylenic unsaturation 11 wherein:

any one of the W groups can be X*;

--X*, A*, and n have been defined previously;

W is alkyl of from 1 to 3 carbon atoms, e.g., methyl;

W is hydrogen, hydroxy, oxo or acetoxy;

W is hydrogen or hydroxyl;

W is hydrogen, hydroxyl or taken together with W oxy (-O-); and

W is hydroxy, acetoxy, or alkoxy of from 1 to 3 carbon atoms;

Those preferred compounds having the basic morphine structure will have the following formula:

wherein:

one of W and W is -X**;

when other than X**; W is methyl; and W is hydrogen, methyl, acetyl or glucuronyl; W is hydrogen or acetyl, usually hydrogen; X** is wherein Z is hydrocarbylene of from 1 to 7 carbon atoms, preferably aliphatic, having from 0 to 1 site of ethylenic unsaturation; and Z** is an enzyme, either specifically labelled with n equal to 1 to 2 ligands or randomly (random as to one or more particular available reactive functionalities) labelled 'with n equal to 2 to 30, more usually 2 to 20, the enzyme retaining a substantial proportion of its activity. The enzyme will be of from about 10,000 to 300,000, frequently about 10,000 to 150,000 molecular weight and is preferably an oxidoreductase, e.g., malate dehydrogenase, lactate dehydrogenase, glyoxylate reductase, or glucose 6-phosphate dehydrogenase, or a glycosidase, e.g., lysozyme or amylase.

Illustrative opiates which can be bound to an enzyme include morphine, heroin, hydromorphone, oxymorphone, metopon, codeine, hydrocodone, dihydrocodeine, dihydrohydroxycodeinone, pholcodine, dextromethorphan, phenazocine, and dionin and their metabolites.

Preferred compounds have W or W as -X*A* or have W and W taken together to provide Methadone Another group of compounds having narcotic activityis methadone and its analogs, which for the most part have the following formula: v

wherein any one of the W groups can be X*;

X*, A*, and n have been defined previously;

p is 0 or 1, usually being the same in both instances;

q is 2 or 3;

W is hydrogen;

W and W are hydrogen, alkyl 'of from 1 to 3 carbon atoms, e.g., methyl, or may be taken together to form a six-membered ring with the nitrogen atom to which they are attached, e.g., pentylene-1,5 and 3-oxa or 3- azapentylene lj; I

W is hydrogen or methyl, only one W W is hydrogen;

W is hydrogen or hydroxyl;

W is hydrogen, acyloxy of from 1 to 3 carbon atoms, e.g., propionoxy, or hydroxy (when W and W are both hydroxy, the oxo group is intended); and

W is hydrogen or alkyl of from 1 to 3 carbon atoms,

e.g., ethyl.

{illustrative compounds which can be linked to an enzyme are methadone, dextromoramide, dipipanone, phenadoxone, propoxyphene (Darvon) and acetylmethadol.

Metabolites of methadone and methadone analogs are also included. Among the metabolites for methadone is N- methyl 2-ethyl-3,3-diphenyl-S-methylpyrroline.

Preferred compounds are when W or W is -'X*.

A narrower class of methadone and its analogs are 0 the formula:

being methyl;

any one of the W groups can be X*; X*, A* and n have been defined previously; I W and W are hydrogen;

13 W and W are methyl or are taken together with the nitrogen atom to which they are attached to form a morpholino or piperidine ring; W and W are hydrogen, hydroxy, acetoxy, at least one being hydroxy or acetoxy; and W"' is alkyl of from 1 to 3 carbon atoms.

The methadone derivatives will for the most part have the following formula:

wherein one of W or W is X**; X**, A**, and n have been defined previously; is phenyl; when otherthan X** W is methyl; and W is propyl.

The metabolites of methadone and close analogs will for the most part have the following formula:

wherein:

any one of the W groups can be -X*, X*, A* and n have been defined previously;

45 is phenyl;

W is hydrogen, hydroxyl, methoxyl or acetoxyl, that is of from 0 to 2 carbon atoms, and except when hydrogen of from 1 to 2 oxygen atoms;

W is hydrogen, methyl, or a free valence joined with Wlslll;

W is an unshared pair of electrons;

W is hydrogen or methyl;

W is hydrogen, hydroxy, or taken together with W forms a double bond between the nitrogen atom and the carbon atom to which W and W are respectively attached; and

W is alkyl of from one to three carbon atoms, usually two carbon atoms, or may be taken together with W to form alkylidenyl of from 1 to 3 carbon atoms, usually 2 carbon atoms.

Preferred compounds are those where W or W are X*, particularly W with W as methyl.

Illustrative compounds which may be linked to an enzyme include phenylbenzyl( l-dimethylamino-Z-propyl methyl succinate,

phenylbeuzyl( l-dimethylamino-Z-propyl) methyl oxalate,

diphenyl (Z-dimethylaminol-propyl) methyl maleate,

O-carb oxymethyl-4,4-diphenyl-7dimethylamino-Z- heptanone oxime,

'4,4-diphenyl-7-dimethylamino-3-octyl succinate,

N-( 2,2-diphenyl-3 -methyl-4-morpholinobutyryl) glycine,

3-ethyl-4,4-diphenyl-6-dimethylaminohept-2-enoic acid,

6-keto-7,7-diphenyl-9-diphenyl-9-(dimethylamino) decanoic acid,

N-carboxymethyl 2-ethyl-3,3-diphenyl-5- methylpyrrolidine.

1 4 'Meperidine The third group of compounds which have narcotic activity and are meperidine or meperidine analogs, have for the most part the following formula:

wherein:

any one of the W groups can be ---X*;

X*, A*, and n have been defined previously;

W is hydrogen;

W is hydrogen, alkyl of from 1 to 3 carbon atoms, e.g., methyl, amiophenylalkyl, e.g., 5- (p-aminophenyDethyl, or phenylaminoalkyl, e.g., phenylaminopropyl, (alkyl of from 2 to 3 carbon atoms);

W is alkoxy of from 1 to 3 carbon atoms, e.g., ethoxy;

and

W is hydrogen or methyl.

Illustrative compounds are meperidine, alphaprodine, alvodine and anileridine.

Preferred compounds are those where W or W is X* or a hydrogen of W is replaced with X*.

Indole alkaloids A second group of ligands of interest are based on tryptamine and come within the class of indole alkaloids, more specifically ergot alkaloids. These compounds will have the following minimal structure:

wherein m, X and A have been defined previously.

Other groups of alkaloids include the steroid alkaloids, the iminazolyl alkaloids, the quinazoline alkaloids, the isoquinoline alkaloids, the quinoline alkaloids, quinine being the most common, and the diterpene alkaloids.

For the most part, the alkaloids bonded to an enzyme will be of from about 300 to 1,500 molecular weight, more usually of from about 400 to 1,000 molecular weight. They are normally solely composed of carbon, hydrogen, oxygen, and nitrogen; the oxygen is present as oxy and oxo and the nitrogen present as amino or amido.

Catecholamines was wherein any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydrogen or alkyl of from 1 to 3 carbon atoms, e.g.,

methyl;

W is hydrogen, or alkyl of from 1 to 3 carbon atoms,

e.g., methyl;

W and W are hydrogen;

W is hydrogen, hydroxy, dimethoxycarboxyphenacyl,

and dimethoxy-a-phthalidyl;

W and W are hydrogen, one of which may be taken with W to form a bond, and when W and W are taken together, each of W and W and W and W may be taken together to form a double bond;

W is hydrogen or alkoxy of from 1 to 3 carbon atoms;

e.g., methoxy;

W and W are hydroxy or alkoxy of from 1 to 3 carbon atoms, e.g., methoxy.

Illustrative compounds include cotainine, narceine, noscapine and papaverine.

Preferred compounds are where W", W or W are X* or have a hydrogen replaced with X*.

A group of compounds related to the catecholamines are epinephrine, amphetamines and related compounds. These compounds have the formula:

wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W and W are hydrogen or alkyl of from 1 to 3 carbon atoms, e.g., methyl and isopropyl, preferably one is hydrogen;

W is hydrogen, alkyl of from 1 to 3 carbon atoms, e.g., methyl and ethyl, or may be taken together with W to form a ring having six annular members with the nitrogen as the only heteroatom;

W is hydrogen, hydroxyl, carbomethoxy, or may be taken together with W to form a morpholine ring;

W is carbomethoxy, when W and W are taken to gether to form a piperidine ring; and

W and W are hydrogen, hydroxyl or alkoxyl of from 1 to 3 carbon atoms.

Illustrative compounds which can be bonded to an enzyme are ephedrine, epinephrine, L-dopa benzidrine (amphetamine), paredrine, methamphetamine, methyl phenidate and norephedrine.

Illustrative compounds which can be linked to an enzyme include 3-(3',4'-dihydroxypheny1) -3-hydroxypropionic acid, N- )3- )3, 3 ,4 trihydroxyphen) ethyl) N-methyl glycine, N 1-phenyl-2-propyl) oxalamic acid.

-( 1-phenyl-2-methylamino-l-propyl) glycolic acid, p-(Z-methylaminopropyH phenoxyacetic acid,

N- l'-phenyl-2'-propyl) glycine, 4-methylamino-4-phenylvaleric acid,

para- (Z-aminopropyl- 1 phenoxyacetic acid,

1 6 4-methylamino-5-phenylvaleric acid, and 3-amino-4-phenylbutyric acid.

Where W and W are hydrogen, preferred compounds will have the following formula:

in 43 W42 any one of the W groups can be --X*;

X*, A* and n have been defined previously;

W and W are hydrogen or alkyl of from 1 to 3 car'- bon atoms, preferably one is hydrogen;

W is hydrogen, -methyl or may be taken together with W to form a piperidine ring;

W is hydrogen, hydroxyl or carbomethoxy; and

W is hydrogen.

Where W and W are oxy, the preferred compounds have the following formula: j

W43 W42" JJH- HN wherein any one of the W groups can be X*;

X*, A* and n have been defined previously; W W and W are hydrogen or methyl; W is hydrogen or hydroxyl; and

W and W are hydroxyl or methoxyl.

Closely related compounds to the amphetamines are those where a saturated five or six membered ring is substituted for the phenyl ring. These compounds will have the following formula:

any one of the W groups is -X*; W have been defined above; W is hydrogen or methyl;

W is hydrogen or hydroxyl; W is hydrogen; and

b is an integer of from four to five.

Of particular interest are those amphetamines bonded to enzymes of the following formula:

wherein one of W W and W is --X**; when other than -X** Barbiturates A wide class of synthetic drugs which finds extensive and frequent abuse are the barbiturates. These compounds are synthetically readily accessible and their use only dif 1 7 ficultly policed. The compounds which find use will com within the following formula:

wan

WEL'IL w 51 any one of the W groups can be -X*;

X*, A*, and n have been defined previously;

W is hydrogen, alkyl of from 1 to 3 carbon atoms, e.g.,

methyl or alkali metal, e.g., sodium;

W and W are hydrogen, alkyl, alkenyl, cycloalkyl, cy-

cloalkenyl, or aryl hydrocarbon of from 1 to 8, more usually 1 to 6 carbon atoms, e.g., ethyl, n-butyl, u-methylbutyl, isoamyl, allyl, A -cyclohexenyl, and phenyl;

W is hydrogen, or alkali metal, e.g., sodium;

W is oxygen or sulfur.

wherein:

Of particular interest are those barbiturates bonded to an enzyme of the formula:

0 ts, \g was wherein one of W and W is X**; when other than -X**:

W is hydrogen, methyl or alkali metal, e.g., sodio; and

W is hydrocarbon of from 1 to 8 carbon atoms, having from 0 to 1 site of ethylenic unsaturation;

W is hydrocarbon of from 2 to 8 carbon atoms, having from 0 to 1 site of ethylenic unsaturation;

is ZCO, wherein Z is hydrocarbylene of from 1 to 7 carbon atoms, usually aliphatic, having from 0 to 1 site of ethylenic unsaturation;

A** and n have been defined previously.

Glutethimide Another compound of interest is g-lutethimide, wherein the enzyme bound analog will have the following formula:

Wa t! wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W and W are hydrogen; and

W' is lower alkyl of from 1 to 3 carbon atoms, e.g.,

ethyl.

Cocaine A drug of significant importance in its amount of use is cocaine. The enzyme bound cocaine or cocaine metabohtes or analogs, such as ecgonine, will for the most part have the following formula:

CHO OW JJH-OW) A CHz- 0 wherein: l 1' any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydroxy, methoxy, amino or methylamino;

W is hydrogen or benzoyl; and

W is hydrogen or alkyl of from 1 to 3 carbon atoms, e.g.,

methyl.

Of particular interest are those ecgonine derivatives (including cocaine derivatives) of the formula:

- c ow N-W -ow A" wherein one of W and W is --X*; when other than W is hydrogen or benzoyl; and W is methyl;

W is hydroxy or methoxy; X** is wherein Z is methylene or carbonyl; or-Z-CO wherein Z is hydrocarbylene of from 1 to 7 carbon atoms, usually aliphatic, having from 0 to 1 site of ethylenic unsaturation;

A** and n have been defined previously.

Diphenyl Hydantoin Another compound of interest is the antiepileptic drug diphenyl hydantoin. This compound and its analogs will have the fol-lowing formula:

wherein any one of the W groups can be --X*;

X*, A* and n have been defined previously; is phenyl;

W W and W are hydrogen.

Marijuana Because of its ready availability and widespread use, tetrahydrocannabinol (the active ingredient of marijuana) and its congeners, cannabidiol and cannabinol and their metabolites are compounds of great interest, where a simple assay method would be of importance. The comformula wall) -3 sites of ethylenie unsaturation,

(particularly A", A" and A wherein any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W is hydrogen or carboxyl;

W is hydroxyl or methoxyl;

W is hydrogen;

W is pentyl or hydroxypentyl;

W is hydrogen, methyl, or the two W s may be taken together to form a carbocyclic ring of from to 6 annular members; and

W is methyl, hydroxymethyl or carboxyl.

Tranquilizers A number of compounds have tranquilizer effects and because of their misuse or abuse do provide opportunities where the determination could be of use.

The first tranquilizer of interest is Meprobamate, also known as Miltown or Equanil. This compound and related analogs have the following formula:

wherein:

any one of the W groups can be -X*; X*, A* and n have been defined previously; W and W are amino.

The next group of tranquilizers are benzdiazocycloheptanes and are known as Librium, Valium, Diazepam, or Oxazepam. These compounds and their related analogs will have the following formula:

Wail was: W080 I N W m Wull wherein:

20 The next group of compounds are the phenothiazines of which chlorpromazine is a member. These compounds will for the most part have the following formula:

Wm nwsil will wherein any one of the W groups can be -X*;

X*, A*, and n have been defined previously;

W is hydrogen, alkyl of from 1 to 6 carbon atoms, dialkylaminoalkyl of from 4 to 8 carbon atoms, e.g., 3(dimethylamino)propyl; N-hydroxyalkyl (alkyl of from 2 to 3 carbon atoms), N'-piperazinoalkyl (alkyl of from 2 to 3 carbon atoms), e.g., N-hydroxyethyl N'- piperazinopropyl; N-alkyl (alkyl of from 1 to 3 carbon atoms) N-piperazinoalkyl (alkyl of from 2 to 3 carbon atoms), e.g., N-methyl N-piperazinopropyl; and 2-(N- alkyl)-piperidinoalkyl, wherein the N-alkyl is of from 1 to 3 carbon atoms and the other alkyl is of from 2 to 3 carbon atoms, e.g., Z-(N-methyl)-piperidinoethyl, there being at least two carbon atoms between the heteroatoms;

W is hydrogen, chloro, trifiuoromethyl, alkylmer'capto of from 1 to 3 carbon atoms, e.g., methylmercapto and acyl of from 1 to 3 carbon atoms, e.g., acetyl; and

W and W are hydrogen.

Amino acids, polypeptides and proteins The next group of compounds are the amino acids, polypeptides and proteins. For the most part, the amino acids range in carbon content from 2 to 15 carbon atoms, and include a variety of functional groups such as mercapto, dithio, hydroxyl, amino, guanidyl, pyrrolidinyl, indolyl, imidazolyl, methylthio, iodo, diphenylether, hydroxyphenyl, etc. These, of course, are primarily the amino acids related to humans, there being other amino acids found in plants and animals.

Polypeptides usually encompass from about 2 to amino acid units (usually less than about 12,000 molecular weight). Larger polypeptides are arbitrarily called proteins. Proteins are usually composed of from 1 to 20 polypeptide chains, called subunits, which are associated by covalent or non-covalent bonds. Subunits are normally of from about 100 to 400 amino acid groups (-10,000 to 50,000 molecular weight).

Individual polypeptides and protein subunits will normally have from about 2 to 400, more usually from about 2 to 300 recurring amino acid groups. Usually, the polypeptides and protein subunits of interest will be not more than about 50,000 molecular weight and greater than about 750 molecular weight. Any of the amino acids may be used in preparing the polypeptide. Because of the wide variety of functional groups which are present in the amino acids and frequently present in the various naturally occurring polypeptides, the enzyme bonded compound can be bonded to any convenient functionality. Usually, the enzyme bonded compound can be bonded to a cysteine, lysine or arginine, tyrosine or histidine group, although serine, threonine, or any other amino acid with a convenient functionality, e.g., carboxy and hydroxy, may be used.

For the most part, the enzyme-labeled polypeptides will have the following formula:

an amino acid residue, r being an integer of from 1 to 1,000, more usually of from 1 to 500, and most com 21 monly of from 2 to 100. r is an integer of at least one and not greater than the molecular weight of the polypeptide divided by 2,000.

Illustrative amino acids include glycine, alanine, serine, histidine, methionine, hydroxyproline, tryptophan, tyrosine, thyroxine, ornithine, phenylalanine, arginine, and lysine. Polypeptides of interest are ACTH, oxytocin, lutenizing hormone, insulin, Bence-Iones protein, chorionic gonadotropin, pituitary gonadotropin, growth hormone, rennin, thyroxine bonding globulin, bradykinin, angiotensin, follicle stimulating hormone, etc.

In certain instances, it will be desirable to digest a protein and assay for the small polypeptide fragments. The concentration of the fragment may then be related to the amount of the original protein.

Steroids Another important group of compounds which find use in this invention are the steroids, which have a wide range of functionalities depending on their function in the body. In addition to the steroids, are the steroidmimetic substances, which while not having the basic polycyclic structure of the steroid, do provide some of the same physiological effects.

The steroids have been extensively studied and derivatives prepared which have been bonded to antigenic proteins for the preparation of antibodies to the steroids. Illustrative compounds include: 17B-estradiol-6-(O-carboxymethyl-oxime)-BSA (bovine serum albumin) (Exley, et al., Steroids 18 593, (1971); testosterone-3-oxime derivative of BSA (Midgley, et al., Acta Endocr. 64 supplement 147, 320 (1970) and progesterone-3-oxime derivative of BSA (Midgley, et al., ibid.)

For the most part, the steroids used have the following formula:

wherein m, X and A have been defined previously. Usually, the enzyme will be bonded to the A, B, or C rings, at the 2, 3, 4, 6 or 11 positions, or at the 16 or 17 positions of the D ring or on the side chains at the 17 position. Of particular interest is where X is bonded to the 6 position. The rings may have various substituents, particularly methyl groups, hydroxyl groups, oxocarbonyl groups, ether groups, and amino groups. Any of these groups may be used to bond the enzyme to the basic ring structure. For the most part, the steroids of interest Will have at least one, usually 1 to 6, more usually -1 to 4 oxygen functionalities, e.g., alcohol, ether, esters, or keto. In addition, halo substitutents may be present. The steroids will usually have from 18 to 27 carbon atoms, or as a glycoside up to 50 carbon atoms.

The rings may have one or more sites of unsaturation, either ethylenic or aromatic and may be substituted at positions such as the 6, 7 and 11 positions with oxygen substituents. In addition, there may be methyl groups at the and 13 positions. The postion marked with a Z, 17, may be and will be varied widely depending on the particular steroid. Z represents two monovalent groups or one divalent group and may be a carbonyl oxygen, an hydroxy group, an aliphatic group of from 1 to 8 carbon atoms, including an acetyl group, an hydroxy-acetyl group, carboxy or carboxyalkyl of from 2 to 6 carbon atoms, an acetylenic group of from 2 to 6 carbon atoms or halo substituted alkyl or oxygenated alkyl group or a group having more than one functionality, usually from 1 to 3 functionalities.

For the second valence of Z, there may be a H or a second group, particularly hydroxyl, alkyl, e.g., methyl, hydroxyalkyl, e.g., hydroxymethyl; halo, e.g., fluoro or chloro, oxyether; and the like.

These steroids find use as hormones, male and female (sex) hormones, which may be divided into oestrogens, gestrogens, antrogens, a-drenocortical hormones (glucocorticoids), bile acids, cardiotonic glycosides and aglycones, as well as saponins sapogenins.

Steroid mimetic substances, particularly sex hormones are illustrated by diethyl stilbestrol.

The sex hormones of interest may be divided into two groups; the male hormones (androgens) and the female hormones (oestrogens).

The androgens which find use will have the following formula:

f woo g 0-1 site of ethylenic unsaturation J wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydrogen, or hydroxyl;

W is hydrogen, methyl or hydroxyl (when two groups bonded to the same carbon atom are hydroxyl, oxo is intended);

W and W are hydrogen or hydroxyl, at least one of W is hydroxy (either as hydroxy or oxo);

W is hydrogen, or two W s may be taken together to form a double bond;

W is methyl; and

W is hydrogen.

win we o-1 site of ethylenie unsaturation w7a wu,

wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W and W are hydrogen, ethinyl or hydroxyl (when two hydroxyls are bonded to the same carbon atom, oxo is intended);

W is hydrogen or hydroxyl;

23 W" is hydroxyl or alkoxyl of from 1 to 3 carbon atoms; W" is hydrogen or two W s may be taken together to form a double bond; and W is hydrogen.

Illustrative compounds which may be bonded to an enzyme are equilenin, fi-estradiol, estrone, estriol, and 17- a-ethinyl-estradiol.

The oestrogens have an aromatic A ring and for the most part have the following formula:

wherein any one of the W groups can be X*;

X*, A* and n have been define-d previously;

W" and W are hydrogen, ethinyl or hydroxyl (when two hydroxyls are bonded to the same carbon atom, oxo is intended);

W is hydrogen or hydroxyl;

W is hydroxyl or al'koxyl of from 1 to 3 carbon atoms;

W is hydrogen or two W s may be taken together to form a double bond; and

W" is hydrogen.

Illustrative compounds which may be bonded to an enzyme are equilenin, ,B-estradiol, estrone, estriol, and 17-a-ethinyl-estradiol.

Illustrative compounds which may be linked to an enzyme include 3-carboxymetl1yl estradiol, 2-chloromethylestrone, estrone glutarate, O-carboxymethyloxime of 6-1ketoestradiol, equilenyl N-carboxymethyl thiocarbamate.

Another class of hormones are the gestogens which have the following formula:

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W and W are hydrogen or hydroxyl, at least one being hydroxyl (where two hydroxyl groups are bonded to the same carbon atom, oxo is intended);

W is hydrogen or hydroxyl;

W and W are hydrogen or hydroxyl, at least one being hydroxyl; and

W is hydrogen, or two W s may be taken together to form a double bond.

Illustrative compounds which may be bonded to an enzyme include progesterone, pregnenolone, allopregnane- 3az20a-diol and allopregnan-3a-ol-20-one.

Illustrative compounds which may be linked to an enzyme include -progesterone O-carboxymethyl oxime, pregn-4-en-20-0n-3ylidinylmethylenecarboxylic acid, O-carboxymethyl progesterone 3-oxime, pregnenolonyl tartrate,

O-pregnenolonyl tartrate,

O-pregnenolonyl lactic acid, and allopreganane-3-carboxyrnethyl-20-ol.

24 The next important group of steroids is the corticosteroids which includes both the mineralcorticoids and the glucocorticoids. These compounds have the following formula:

\i/ Wv5 wan ivw won 0-1 site of ethylenic unsaturation J I1 wherein any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydrogen or hydroxyl;

W and W are hydrogen or hydroxyl, at least one of which is hydroxyl (when two hydroxyl groups are bonded to the same carbon atom, oxo is intended);

W is hydrogen or hydroxyl;

W W W and W are hydrogen or hydroxyl, at

least one of W and W is hydroxyl;

W is methyl or formyl; and

W is hydrogen or two W s may be taken together to form a double 'bond.

0-1 site of ethylenlc unsaturatlon o 0 W I (L wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W W W and W are hydrogen, hydroxy], or a glycoside, at least one being hydroxyl or a sugar, mostly as a glycoside. The sugars include xylose, glucose, cymarose, rhamnose, and galactose.

Also of interest are the saponins and sapogenins derived from plants. These compounds have a spiro ring structure at C22.

Sugars The next group of compounds are the sugars and saccharides are combinations of various sugars to form dimers, tn'mers and high molecular weight polymers, referred to as polysaccharides.

Prostaglandin Another group of compounds of biological importance are the prostaglandins. These compounds when bonded to enzymes have for the most part the following formula:

-1 site of ethylenic unsaturation Cs o-n 01 site of ethylenic unsaturation wherein any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydrogen or hydroxyl;

W and W are hydrogen or hydroxyl, (where two hydroxyl groups are bonded to the same carbon atom, oxo is intended);

W is hydrogen or hydroxyl; and

W" is hydroxyl, amino or an oxy group of from 1 to 6 carbon atoms, e.g., alkoxy.

Miscellaneous Included is this group are the antibiotics such as penicillin, chloromycetin, actinomycetin, tetracycline, terramycin, and nucleic acids or derivatives, such as nucleosides and nucleotides.

Also of interest is serotonin which is 3-(2'-aminoethyl)-5-hydroxyindole. X* may be bonded at either of the amino nitrogen atoms or the hydroxyl group.

Of course, many of the compounds which are of interest undergo metabolic changes, when introduced into a vertebrate. The particular physiological fluid which is tested may have little, if any of the original compound. Therefore, the original presence of the compound might only be detectable as a metabolite. In many instances, the metabolite may be the glucuronide, either oxy or 0x0 derivative of the original compound. In other instances, the original compound may have undergone oxidation, e.g., hydroxylation, reduction, acetylation, deamination, amination, methylation or extensive degradation. Where the metabolite still retains a substantial portion of the spatial and polar geometry of the original compound, it will be frequently possible to make the ligand analog based on either the original compound or metabolite. Where the metabolite is distinctively different than the original compound, the ligand analog will be based on the metabolite.

Of particular interest as metabolites, particularly of the steroids, are the sulfates and glucuronides.

Besides meabolites of the various drugs, hormones and other compounds previously described, of significant interest are metabolites which relate to diseased states. Illustrative of such compounds are spermine, galactose,

26 phenylpyruvic acid and porphyrin Type 1, which are believed to be diagnostic of certain tumors, galactosemia, phenylketonuria and congenital porphyra, respectively.

Two compounds of interest which are metabolites of epinephrine are vanillylmandelic acid and homovanillic acid. With these compounds, either the hydroxyl or carboxyl groups can be used as the site for X*.

Another general category of interest is the pesticides, e.g., insecticides, fungicides, bacteriocides and nematocides. Illustrative compounds include phosphates such as malathion, DDVP, dibrom; carbamates, such as Sevin, etc.

Since many of the biologically active materials are active in only one stereoisomeric form, it is understood that the active form is intended or the racemate, where the racemate is satisfactory and readily available. The antibodies will be specific for Whatever form is used as the hapten.

Enzymes (A) Enzymes vary widely in their substrates, cofactors, specificity, ubiquitousness, stability to temperature, pH optimum, turnover rate, and the like. Other than inherent factors, there are also the practical considerations, that some enzymes have been characterized extensively, have accurate reproducible assays developed, and are com mercially available. In addition, for the purposes of this invention, the enzymes should either be capable of specific labelling or allow for efiicient substitution, so as to be useful in the subject assays. By specific labelling is intended selective labelling at a site in relationship to the active site of the enzyme, so that upon bindin of the receptor to the ligand, the enzyme is satisfactorily inhibited. By allowing for efiicient substitution to be useful in the subject assay, it is intended that the enzyme be inhibited sufliciently when the ligand is bound to the receptor, and that the degree of substitution required to achieve this result does not unreasonably diminish the turnover rate for the enzyme nor substantially change the enzymes solubility characteristics.

From the standpoint of operability, a very wide variety of enzymes can be used. But, as a practical matter, there will be a number of groups of enzymes which are preferred. Employing the International Union of Biochemists (I.U.B.) classification, the oxidoreductases (1.) and the hydrolases (3.) will be of greatest interest, while the lyases (4.) will be of lesser interest. Of the oxidoreductases, the ones acting on the CHOH group, the aldehyde or keto group, or the CH-NH group as donors (1.1, 1.2, and 1.4 respectively) and those acting on hydrogen peroxide as acceptor (1.l1) will be preferred. Also, among the oxidoreductases as preferable will be those which employ nicotinamide adenine dinucleotide, or its phosphate or cytochrome as an acceptor, namely 1.x.l and 1.x.2, respectively under the I.U.B. classification. Of the hydrolases, of particular interest are those acting on glycosyl compounds, particularly glycoside hydrolases, and those acting on ester bonds, both organic and inorganic esters, namely the 3.1 and 3.2 groups respectively, under the I.U.B. classification. Other groups of enzymes which might find use are the transferases, the lyases, the isomerascs, and the ligases.

In choosing an enzyme for commercialization, as compared to a single or limited use for scientific investigation, there will be a number of desirable criteria. These criteria will be considered below.

The enzyme should be stable when stored for a period of at least three months, and preferably at least six months at temperatures which are convenient to store in the laboratory, normally 20 C. or above.

The enzyme should have a satisfactory turnover rate at or near the pH optimum for binding to the antibody, this is normally at about pH 6-10, usually 6.0 to 8.0. Preferably, the enzyme will have the pH optimum for the 27 28 turnover rate at or near the pH optimum for binding of to 150,000 molecular weight, and frequently from 10,000 the antibody to the ligand. to 100,000 molecular weight. Where an enzyme has a A product should be either formed or destroyed as a plurality of subunits the molecular weight limitations result of the enzyme reaction which absorbs light in the refer to the enzyme and not to the subunits. ultraviolet region or the visible region, that is the range For synthetic convenience, it is preferable that there of about 250-750 mm, preferably 300-600 nm. be a reasonable number of groups to which the ligand Preferably, the enzyme should have a substrate (includmay be bonded, particularly amino groups. However, ing cofactors) which has a molecular weight in excess of other groups to which the ligand may be bonded include 300, preferably in excess of 500, there being no upper hydroxyl groups, thiols, and activated aromatic rings, e.g., limit. The substrate may either be the natural substrate, phenolic. or a synthetically available substrate. Therefore, enzymes will preferably be chosen which are Preferably, the enzyme which is employed or other sufiiciently characterized so as to assure the availability enzymes, with like activity, will not be present in the of sites for linking, either in positions which allow for fluid to be measured, or can be easily removed or deactiinhibition of the enzyme when the ligand is bound to vated prior to the addition of the assay reagents. Also, antibody, or there exist a sufficient number of positions one would Want that there not be naturally occurring as to make this occurrence likely. inhibitors for the enzyme present in fluids to be assayed. A list of common enzymes may be found in Hawk,

Also, although enzymes of up to 600,000 molecular et 211., Practical Physiological Chemistry, McGraw-I-Iill weight can be employed, usually relatively low molecular Book Company, New York (1954), pages 306 to 307. weight enzymes will be employed of from 10,000 to That list is produced in total as follows, including the 300,000 molecular weight, more usually from about 10,000 source of the enzyme, the subtrate and the end products.

Name and class Distribution Substrate End-products Hydrolases carbohydrascs: Carbohydrates 1. Amylase Pancreas, saliva, malt, etc Starch, dextrin, etc Maltese and dextriris. 2. Lactase-. Intestinal Juice and mucosa. Lactose Glucose and galactose. 3. Maltase lntestinaljuice, yeast, etc Maltose. Glucose. 4. Sucr do Sucrose". Glucose and fructose. 5. Emulsin Plants B-Glucosides Glucose, etc.

N ucleases: Nucleic acid and derivatives 1. Polynuclcotidase Pancreatitciuice, intestinal Nucleic acid Nucleotides.

Juice, e -c. 2. Nucleotidase Inttestinaliuice and other Nucleotides Nucleotides and phosphoric acid.

ISSUES. 3. Nucleotidasc Animal tissues .do Carbohydrate and bases.

Amidases: Amino compounds and amides 1. Argiiiase Liver Arginine Ornithine and urea. 2. Urease Ba cteria, soybean, Jack bean, Urea Carbon dioxide and ammonia.

e c. 3. Glutairiinase Liver, etc Glutamine Glutamic acid and ammonia. 4. Transamiuase Animal tissues Glutamic acid and oxalacctic acid, e-Ketoglutaric acid, aspartic acid, ctc.

Purine deaminases: Purine bases and derivatives 1. Adenase- Animal tissues Adenine.. Hypoxanthinc and ammonia. 2. Guanase ..do Guanine- Xautliiiic and ammonia.

Pcptidases: Peptides 1. Aminopolypeptidase. Yeast, intestines, etc Polypeptides Simpler peptides and amino acids. 2. Carboxypeptidase Pancreas. .do Do. 3. Dipeptidase Pllginttar d animal tissues and Dipeptides Amino acids.

6H3. 4. Prolinase Animal tissues and yeast Proline peptides Proline and simpler peptides.

Proteinascs: Proteins 1. Pepsin Gastn'el'uice Pro Pro P peptones, etc. 2. Trypsin Pancreatic Juice Proteins, proteoses, and peptones Polypeptides and amino acids. 3. Cathepsi Animal tissues Protein Proteoses, and peptones. 4. Rennin- Calf stomach Casein- Paracasein. 5. Chymotrypsin Pancreatic juice Proteins, proteosm and peptones Polypeptides and amino acids. 6. Papain Papaya, other plants do 7. Ficin Fig sap Proteins Proteoses, etc.

Esters Alcohols and acids Esterases:

1. Li ase Pancreas, castor bean, etc Fats Glycerol and fatty acids. 2. Esterases.... Liver, etc Ethyl butyrate, etc--. Alcohols and acids. 3. Phosphatases. Plant and animal tissues Esters of phosphoric acid.- Phosphate and alcohol. 4. Suliatases Animal and plant tissues Esters of sulfuric acid"--. Sulfuric acid and alcohol. 5, Cholinosfarase Blood, tissues Acetylchcline Choline and acetic acid. Iron enzymes:

1. Catalase All living organisms except a Hydrogen peroxide Water and oxygen.

few species of microorganisms. 2. Cytochrome nridase do Reduceifi cytochrome C in the pres- Oxidized cytochrome C and water.

ence 0 oxygen. 3. Peroxidase Nearly all plant cells A large number of phenols, aromatic Oxidation product of substrate and amines, etc. in the presence of H202. wa er. Copper enzymes:

1. Tyrosinase (poly-phcnol- Plant and animal tissues Various phenohe compounds Oxidation product of substrate.

oxidgse, monoplienoloxidase 2. Ascorbic acid oxidase Plant tissues Ascorbic acid in the presence of oxygen. Dehydroascorbic acid. Enzymes containing coenzymes I and/or II:

1. Alcohol dehydrogenase Animal and plant tissues Ethyl alcohol and other alcohols Acetaldehyde and other aldchydcs. 2. Malic dehydrogenase.-.- do... L( malic acid OXalacetic acid 3. Isocitric dehydrogenasedo.-- L-isocitric acid..-- Oxalosuccinic acid. 4. Lactic dehydrogeiiase Animal tissues and ye Lactic acid Pyruvic acid. 5. fi-Hydrorylbutyric dehy- Liver, kidneys, and heart L-B-hydroxybuty'ric acid Acetoacetic acid.

drogenase. 6. Glucose dehydrogenase... Animal tissues D-glucose D-gluconic acid.

TABLE-Continued Name and class Distribution Substrate End-products 7. Bobison ester dehydrog- Erythrocytes and yeast Robisonester(hexose-fi-phosphate) Phosphohexonic acid.

8119.39. 8. d(Illycerophosphate dehy- Animal tissues Glycerophosphate Phosphoglyceric acid.

ogenase. 9. Aldehyde dehydrogenase... Liver Aldehydes Acids. Enzymes which reduce cytochrome:

1. Succinic dehydrogenase Plants, animals and microor- Succinic acid Fuman'c acid.

(as ordinarily prepared). ganisms Yellow enzymes:

1. Warburgs old yellow Yeast Reduced coenzyme Il Oxidized coenzyme II and reduced yellow enzyme. enzyme. 2. Diaphorase Bacteria, yeasts, higher plants, Reduced coenzyme 1 Oxidized coenzyme I and redu ed yellow and animals. diaphorase. 3. Haas enzy Yeast. Reduced coenzyme II Oi1ridized coenzyme II and red d yel- OW enzyme. 4. Xanthine oxidase Animal tissues Hypoxanthine xanthine, aldehydes, Xanthine, uric acid, acids, oxidized reduced coenzyme I, etc. enzyme I, etc. in presence of air, H201. 5. D-amino acid mri .do D-amino acids plus Oz a-Keto-acids plus NH: plus H 0 6. L-amino acid oxidases Animals, snake venoms Iramino acids Keto acids and ammonia. 7. TPN-cytochrome 0 Yeast, liver Reduced coenzyme II and cytochrome Oxidized coenzyme I and reduc d cytoreductase. C. chrome C. 8. DPN cytochrome 0 Liver, yeast Reduced coenzyme I and cytochrome Do.

reductase. O. Hydrases:

1. Fumarase Living organisms in general Fumanc acid plus 1120 L-malic acid. 2. Aconitase Animals and plants Citric acid cis-Aconitic acid and L-isocitrie id, M 3. Enolase Animal tissues and yeast 2-phosphoglycen'c acid Phospyruvic acid plus H 0,

utases:

1. Glyoxalase Living organisms in general"-.. Methyl gllyoxal and other substituted D lactic acid.

g yoxa Desmolases:

1. Zymohexase (aldolase)-.. All cells Fructose-ldrdlphosphate Dihydroxyacetone phosphoric a id and phosphoglycen'c acid. 2. Carboxylase Plant tissues Pyruvic acid Acetaldehyde and C0 3. fl-Keto carboxylases Animals, bacteria, plants. B-Keto acids. lz-KQtO acids. 4. Amino acid decarboxylases- Plants, animals, bacteria L-ammo acids Amines and C01. 5. Carbonic anhydrase Erythrocytes Carbonic acld C0: H2O. Other enzymes: I

1. Phosphorylase Animal and plant tissues tarch orglycogen and phosphate Glucose-l-phosphate. 2. Phosphohexoisomeraselu se fi-phosphate Fructose-G-phosphate. 3. Hexokinase Adenvsmctl'lphosphate. Ad nosi r iediphosphate plus glucose-6- p osp ate. 4. Phosphoglucomutase Plant and animals lucos -l-ph sphate Glucose-fi-phosphate.

Of the various enzymes, the following table indicates 3, phospholipase C enzymes of particular interest set forth m accordance 3.2 Acting on glycosyl compounds with the I.U.B. classification. 3.2.1 Glycoside hydrolases 1. Oxidoreductases 1. oz-amylase 1.1 Acting on the CH-OH group of donors 40 4. cellulase 1.1.1 With NAD or NADP as acceptor 17. lysozyme 1. alcohol dehydrogenase 23. fl-galactosidase 6. glycerol dehydrogenase 27. amyloglucosidase 2-6. glyoxylate reductasc 31. B-glucuronidase 27. 'L-lactate dehydrogenase 3.4 Acting on peptide bonds 37. malate dehydrogenase 3.4.2 Peptidyl-amino acid hydrolase 49. glucose 6-phosphate dehydrogenase 1. carboxypeptidase A 17. mannitol 1-phosphate dehydrogenase 3.4.4 Pcptidyl-peptide hydrolase 1.1.2 With cytochrome as an acceptor 5. a-chymotrypsin 3. L-lactate dehydrogenase 10. papain 1.1.3 With 0 as acceptor 3.5 Acting on CN bonds other than peptide bonds 4. glucose oxidase 3.5.1 In linear amides 9. galactose oxidase 5. urease 1.2 Acting on the CH-NH; group of donors 3.6 Acting on acid anhydride bonds 1.4.3 With 0 as acceptor 3.6.1 In phosphoryl-containing anhydridcs 2. L-amino acid oxidase 1. inorganic pyrophosphatase 3. D-amino acid oxidase 4. Lyases 1.6 Acting on reduced NAD or NADP as donor 4.1 Carbon-carbon lyases 1.6.99 With other acceptors diaphorase 4.1.2 Aldehyde lyases 1.10 Acting on diphenols and related substances as 7. aldolase donors 4.2 Carbon-oxygen lyases 1.10.3 With 0 as acceptor 4.2.1 Hydrolases 1. polyphenol oxidase 1. carbonic anhydrase 3. ascorbate oxidasc 4.3 Carbon-nitrogen lyases 1.11 Acting on H 0 as acceptor 4.3.1 Ammonia lyases 1.11.1 3. histidase 6. catalase 7. peroxidase Lmkmg Group (X) 3. H ydrolases The ligand or ligand analog is normally bonded either 3.1 Acting on ester bonds 7 directly to the enzyme, by a single or double bond, or 3.1.1 Carboxylic ester hydrolases preferably to a linking group. For those ligands, which 7. cholinesterasc are haptens, and for which the receptors are antibodies 311.3 Phosphoric monoester hydrolases the ligand will have been bound to a protein for the purl. alkaline phosphatase pose of preparing the antibodies. Since the antibodies will 3.1.4 Phosphoric diester hydrolases recognize that portion of the ligand molecule which ex- 31 tends from the protein, ordinarily the same linking group will be attached on the same site on the ligand, as was used in bonding the ligand to the protein to provide the antigenic substance.

The functional groups which will be present in the enzyme for linking are amino (including guanidino), hydroxy, carboxy, and mercapto. In addition, activated aromatic groups or imidazole may also serve as a site for linking.

Amino acids having amino groups available for linking include lysine, arginine, and histidine. Amino acids with free hydroxyl groups include serine, hydroxyproline, tyrosine and threonine. Amino acids which have free carboxyl groups include aspartic acid and glutamic acid. An amino acid which has an available mercapto group is cysteine. Finally, the amino acids which have activated aromatic rings are tyrosine and tryptophan.

In most instances, the preferred linking group will be the amino group. However, there will be situations with certain enzymes, where one of the other linking groups will be preferred.

The ligand, of course, will have great diversity of functionalities which may be present. In addition, as already indicated, the functionalities which are present may be modified so as to form a different functionality, e.g., keto to hydroxy or an olefin to aldehyde or carboxylic acid. To that extent, the choice of groups for linking to the ligand may be varied much more widely than the choice of groups for linking to the enzyme. In both cases, however, a wide variety of different types of functionalities have been developed, specifically for linking various compounds to proteins and particularly enzymes.

Where a linking group is employed for bonding the ligand to the enzyme, it will be the more frequent procedure to bond the linking group to the ligand to provide an active site for bonding to the enzyme. This may be achieved in a single step or may require a plurality of synthetic steps, including blocking and unblocking the active groups on the ligand, other than the one involved in providing the linking group. The linking groups which are reported hereafter are solely concerned with the bridge bonding the enzyme and the ligand.

Where a linking group is used, there will normally be from one atom to 14 atoms in the chain, more usually from two atoms to 8 atoms in the chain bonding the ligand to the enzyme. Where cyclic structures are involved, the cyclic structure will be equated to the number of atoms providing a similar length to the chain.

The linking group (excluding the atoms derived from the ligand and enzyme), when other than a direct bond is involved, will be of from about 1 to 30 atoms-carbon, hydrogen, nitrogen, oxygen, phosphorous, and sulfur more usually 4 to 20 atoms.

Preferably, the linking group will normally be of from zero to 14 carbon atoms, usually from 1 to 8 carbon atoms and from 1 to 8 heteroatoms, and frequently of from 1 to 8 carbon atoms and from 1 to 4 heteroatoms, which are oxygen, sulfur and nitrogen, more usually oxygen and nitrogen. The most frequent heterofunctionalities present in the linking group are nonoxocarbonyl or thiocarbonyl, amino, imino (oxirne or imidate) diazo,

or oxy.

A group of linking groups are derived from a group having a nonoxocarbonyl functionality and when a second functionality is present, the second functionality may be based on a second nonoxocarbonyl functionality, a haloalkyl, O-substituted hydroxylamine, imino, amino or diazo. The linking group will normally have from 2 to 8 carbon atoms and from 1 to 4 heteroatoms which are usually oxygen and nitrogen (the heteroatoms of the ligand and enzyme are not included in the above range of heteroatoms). Such determination is somewhat arbitrary, so that between a carbon atom of the ligand and a carbon atom of the enzyme, there may be as many 32 as six heteroatoms. The heteroatoms may be part of the linking group chain or branched from the chain, e.g., nonoxocarbonyl oxygen.

One group of linking groups will have from 2 to 6 carbon atoms, more usually 2 to 4 carbon atoms and be an aliphatic non-oxo carbonyl functionality. (Another group of linking groups will have from 2 to 8 carbon atoms and have from 1 to 2 heteroatoms, e.g., oxygen and nitrogen, in the chain, such as amino, oximino, diazo, oxy, and the like.

The following tabulation indicates various linking groups, varying with the functionalities present on the ligand and the enzyme. Except as indicated, the linking group satisfies one to two valences on the ligandand enzyme functional groups to which it is bound.

Ligand amino (NH), or hydroxyl Enzyme amino (-NHz), hydroxyl (-0 or mereapto (-SH) (only primary amino) Ligand oxoearbonyl :0)

Enzyme amino (-NHz), hydroxyl (-OH) or mercapto (-SH) Ligand Enzyme amino (-NHz), hy)droxyl (-OH),

O I. non-0x0 carbonyl (C or mercapto (-SH I Z"--arylene of from 6 to 10 carbon atoms.

Where the enzyme is to be linked through a carboxyl group of the ligand or a linking group bonded to the ligand, either esters or amides will be prepared. The ligand may be bonded to any of the linking groups which are appropriate to provide a link between the ligand and the alcohol or amine group of the enzyme to form the ester or amide group respectively. When the enzyme has an activiated aromatic ring, the ligand may be bonded to an aromatic diazonium salt to provide the desired bridge.

The linking group will be selected in accordance with the following considerations. The bonds formed must be stable under the conditions of the assay. When bonding the ligand through the linking group to the enzyme, the enzyme must retain at least a portion of its activity upon isolation, The enzyme must not prevent binding of the ligand to the receptor. The functionalities should permit some selectivity, so that bonding can be directed to specific' groups or types of groups in both the ligands and enzymes.

A few illustrations of how linking groups may be introduced are considered worthwhile. For example, if the ligand has an amino group, the amino may be bonded to form u-bromo-acetamide. This product may then be used to form a carbon nitrogen bond to an amino acid of an enzyme which has a free amine group, e.g., lysine.

If the ligand has a keto group, the carbonyl may be condensed directly with an amine group of the enzyme, or the O-carboxy methyloxime may be prepared with O- carboxymethyl hydroxylamine. A mixed anhydride, with isob'utyl chloroformate is formed, which may then be 34 used to form the carboxamide with the amino group of the lysine.

Where a carboxyl group is present in the ligand, it may be convenient to react the carboxy group to form the monoamide of phenylenediamine. The resulting compound may then be diazotized to form the diazo salt which may be coupled with tyrosine present in the enzyme.

Another way to form the linking group would be to have an alcohol of a ligand react with succinic anhydride to form the monoester. The free carboxy group can then be activated by preparing the mixed anhydride and be used for reaction with an amine in the enzyme.

With an amino group present on the ligand, this may be reacted with maleic anhydride under forcing conditions to prepare the maleimide. The maleimide may then be combined with cysteine in the enzyme to provide by a Michaels addition the 3-thiosuccinimide.

For poly-functionalized ligands such as proteins it will usually be necessary to use special techniques to prevent the formation of enzymes coupled together which are then bonded to the ligand. Having the two or more enzymes coupled would made inhibition difiicult. Techniques can be employed where one group of a bifunctional reagent can be made unreactive, while the other group reacts with the enzyme or protein ligand. The other group can then be activated to carry out the second stage of linking the protein ligand to an enzyme.

Various bifunctional reagents can be employed. For example, a functionalized diazosulfonate can be used. One of the proteins can be bonded to the functionality and then the modified protein added to the other protein and the diazosulfonate group activated with the light.

While for the most part, the enzyme may be bonded to any convenient position of the ligand, either through a functionality naturally present in the ligand or one introduced synthetically, there are preferred methods of bonding the enzyme to the ligand. First, it should be recognized that the ligand of the enzyme-bound-ligand need not have any biological activity. One is primarily concerned in not disturbing the geometry and polar site relationships of a substantial portion of the ligand molecule. Where the ligand is a hapten, the enzyme will therefore normally be bonded at the same site as was employed for attachment to the protein in the preparation of the antigen. Where the ligand is an intact antigen, several sites may be employed for attachment to one or more enzyme molecules with the obvious limitation that the number of enzyme molecules must not be so great as to prevent binding to the antibody. Where the ligand has a natural receptor other than an antibody, the point(s) of attachment will also be determined primarily by the necessity to preserve strong binding to the receptor.

Furthermore, if one is attempting to assay one of a variety of molecules which are quite similar, for example steroids, but differing in their substituents at the 17 position, one would choose to mark the molecule with the enzyme at a site distant from the distinguishing functionality. Following the steroid analogy, it would frequently be preferable to bond at the 3 position, rather than at the 17 position, since the distinctive portion of the molecule is usually at the 17 position. For the most part, the 3 position is either an alcohol or a ketone, the ketone normally being associated with aliphatic unsaturation. Also, the 6 position is a useful site.

The same or similar consideration will be present with other ligands. For example, with a polypeptide, which has a natural receptor site, one would preferably bond away from the receptor site.

The number of ligands which may be bonded to the enzyme will be limited by the number of available sites for bonding to the enzyme. In most cases this will be the amino groups which are present, but as already indicated, carboxyl, hydroxyl, thiol and activated aromatic rings, e.g., phenolic, are also useful sites.

Various factors will affect the number of ligands which is optimum for a specific enzyme and a specific ligand. Of prime consideration is the number required for obtaining the desired degree of inactivation when receptor is bound to the enzyme-bound-ligand. The number required will vary with the mode of attachment and the conditions for attachment of the ligand to the enzyme. Except under special circumstances, e.g., afiinity labelling, there will usually be differences in degree of inactivation, as to each site to which the receptor is bound to the enzyme through a ligand. In addition, there may be cumulative effects, with an increase in the number of receptors bound to the enzyme through ligand.

Other considerations as to the number of ligands per enzyme will be the effect of the increasing number of ligands on: solubility of the enzyme-bound-ligand; activity of the enzyme-bound-ligand in the absence of receptor; and the sensitivity of the assay. Therefore, the choice of the number of ligands bonded to the enzyme is usually empirically determined, based on the effect of varying the number of ligands on the enzyme has on the assay.

With small enzymes, e.g., lysozyme, those that have molecular weights in the range of 10,000 to 30,000 from 2 to ligands can be sufficient. With larger enzymes, e.g., malate dehydrogenase, of molecular weight in the range of 30,000 to 150,000, 2 to 30 ligands can be sufficient. For malate dehydrogenase 2 to 22 ligands on the average will be employed. As few ligands as possible should be bonded to the enzyme to achieve the desired degree of inhibition. Desirably, the number of ligands per enzyme should be in the range of 1 to 20, more preferably 1 to 12.

As already indicated, because of the diversity of enzymes which can be used for the assay and the variety of functionalities in the enzyme available for attachment, and the varying activities of the functionalities for being bonded to the ligand as well as their relative position to the active site of the enzymes, different numbers of ligands will be necessary for obtaining the desired degree of inhibition, when the enzyme-bound-ligand is bonded to antibody. Furthermore, the desired degree of inhibition may vary, depending on the sensitivity required for an assay for a particular ligand.

It is found, for the most part, that increasing the average number of ligands increases the amount of inhibition, up to a degree of substitution, where further substitution does not provide a significant increase in inhibition. Therefore, by varying the conditions for the reaction between the modified ligand (ligand and linking group) and the enzyme, varying degrees of substitution can be achieved. The time for the reaction, the mole ratio of ligand to enzyme and the like can be varied. Also, the reactive functionality on the linking group can be varied to change the number and sites for substitution. One can then empirically determine the number of ligands required for the desired degree of inhibition.

It should also be noted that in referring to inhibition of an enzyme, the substrate for the enzyme plays a role. Different degrees of inhibition may be achieved with different substrates. Thus, not only can one obtain varying degrees of inhibition by varying the number of ligands bonded to the enzyme, and the sites to which the ligands are bonded, but also, with some enzymes, by varying the substrate for the enzyme.

It is also found that with increasing substitution of the enzyme by ligand, there can be reduction in enzyme activity. The turnover number diminishes and there is a concomitant increase in the Michaelis constant. The decrease in turnover number with increasing substitution will vary with the enzyme. By employing enzymes which have a high initial activity, a loss of as much as seventy-five percent of initial activity can be tolerated.

(Turnover number is the number of substrate molecules transformed per unit time per enzyme molecule. Lehninger, Biochemistry, Worth Publishers, New York, 1970. Since the turnover number is reported at varying temperatures and on varying bases, e.g., weight of protein as an indication of number of enzymes or change in a spectrophotometric value as an indication of number of substrate molecules, there is at the present no simple comparison between the turnover number of different enzymes. Therefore, no minimum numerical turnover number for preferred enzymes can be given.)

Also, the ligand will be attached to the enzyme by a relatively short chain, usually of the order of 1.5 to about 20 A. more usually about 3 to 10 A.

Enzyme Assay Receptor In the subject invention, for the most part, the receptors will be macromolecules which have sites which recognize specific structures. The recognition of the specific structures will be based on Van der Waals forces, which provide a specific spatial environment which maximizes the Van der Waals forces; dipole interactions, either by permanent or induced dipoles; hydrogen and ionic bonding; coordinate covalent bonding; and hydrophobic bonding. For a detailed discussion of mechanisms by which receptors bind ligands, see Goldstein, et al., Principles of Drug Action, Harper and Rowe, New York, 1968. Y

The macromolecules of greatest interest are proteins and nucleic acids which are found in cell membranes, blood, and other biological fluids. These compounds include en zymes, antibodies, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) and natural receptors.

The most convenient group of proteins for use in the subject invention are antibodies. These materials are con-" veniently used in the analysis of the category of ligands referred to as haptens. Antibodies are produced by introducing an immunogenic substance into the bloodstream of a living animal. The response to the introduction of the immunogenic substance for antigen is the production of antibodies which act to coat the antigen and detoxify it or precipitate it from solution. The protein forms a coat which is geometrically arranged so as to have the antigen fit the spatial arrangement of the protein. This maybe analogised to a lock and key. The interaction is normally reversible, in that the antigen is subject to displacement or removal by various means without destruction of the rcceptor site.

There are many materials which are antigens and will produce an immunogenic response by being introduced into the bloodstream of a vertebrate. However, a number" of materials of interest are not antigens, but are haptens,

and in that situation, an extra step in preparing the anti body is required. This method of preparing antibodies with materials other than antigens is well known and may be found in Microbiology, Hoeber Medical Division, Harper and Rowe, 1969. See also, Landsteiner, Specificity of Serological Reactions, Dover Publications, N.Y. 1962; Kabat, et al., Experimental Immunochemistry, Charles C.; Thomas, Springfield, Illinois, 1967; and Williams, et al.,

Methods in Immunology and Immunochemistry, Vol. 1,2"

Academic Press, New York, 1967. r The material which isto be assayed is bonded to protein by any convenient means and the modified protein introduced into the blood stream. The same type of bonding groups used with the enzyme attachment to the ligand may be employed. The antibodies which form will 37' include groupsof antibodies which are shaped to fit the foreign moiety bonded to the protein. Therefore, antibodies are obtained which are specific to the compound or moiety bonded t o'the protein. By careful separation techniques, the antibodies primarily concerned with the 38 pounds of biological interest. Compounds for which receptors are naturally occurring include thyroxine, corticosterone, cortisone, ll-desoxycortisol, ll-hydroxyprogesterone, estrogen, insulin and angiotensin. See, for example, Vonderhaar et al., Biochem. Biophysics Acta.,

5 molety m question, can be concentrated so as to provide 176, 626 (1969). All of these ligands have been studied an antibody composition which is primarily related to the and reported upon in the literature in connection with specific moiety which was bonded to the protein. studies on their binding with specific receptors.

TABLE I Ligand Receptor for ligand reference Ligand structure Thyroxin Thyroxin Binding Globulin (TBG) Thyroxin Binding I I Prealbum (TBA) B.E.P. Murphy, 0. J. .T. Pattee, J. Clin. NH:

Endocn, 24, 187 (1964).

HO- -O -CHz-CH-COOH I Thyroxine Cortieosterone Protein from Brain Cell Nuclei, B. McEwen, L. Plapinger CHzOH Nat. 266, 263 (1970). I

I C=O Corticosterone Cortisol (R=O H, B.E. Murphy, J; Clin. Endocn, 28, 343 (1968), 27, 973 1967) CHzOH Cortisone (R=O) Corticosteroid Binding Globulin (Transcortin). I lkdefixgprtisol 0: T, CHaI Cortisone Estradiol Receptor Site for Estrogen From Uterus, BBA, 176, 626 (1969)- CH '7 i CH 3 U Estradiol Insulin QR. Morgan, W. M. Holland, III Diabetes, 1966 See below} Angiotensin II L.B. Page, E. aber, A.Y. Kimura, A. Peruode, J. 01112. End. See below.

N H; S S NH: N H: N Hg I 1 H-Gly-Ile-V -Glu-Glu-Cys-Cys-Ala-Ser-Val-Cys-Ser-Leu-Tyr-Glu-Leu-Glu-Asp-Tyr-Cys-Asp-OH H-Phe val-Asp-Glu-His-Leu-cys-Gly-S er-His-Leu-Val-G lu-Ala-L eu-T yr-Leu-Val-Cys-Gly-Glu-Ar -G1y-Phe-Phe-Tyr-Thr-Pro-Lys-Ala-0H.

NH: NH: 2 Asp-Arg-Val-Tyr-Ileu-His-Pro-Phe.

To illustrate this method, para-aminobenzene arsonate is diazotized to form the diazo salt. By combining the diazo'salt with rabbit globulin, the rabbit globulin may be labeled with pafa-azobenzene arsonate. By introducing this composition into the blood stream of an animal other than-'a"rabbit, for example, a sheep, antibodies can be formed which will' have a spatial arrangement which accept's solely the a'zobenzene arsonate.

Inaddition to antibodies, there are a number of naturally 'occurringr'e'ceptors which are specific to com- 75 type to evaluate a wide variety of enzymes when bonded to a carboxyl group by means of a mixed anhydride. The information thus obtained can be readily extrapolated to what one would expect from bonding other similar drugs in an analogous manner to the same enzyme.

Experimental The following examples are olfered by way of illustration and not by way of limitation.

(All temperatures are recorded in centigrade.)

INDEX gentle o dcaigboxymethyl morphine conjugate to glyoxylate rec as u e- Ofl-(wisopropyl) carboxymethyl morphine conjugate to malate dehydr e 46 1.9 -carboxymethyl morphine conjugate to glucose 6-phosphate dehydrogenase 47 1.10 0 -imid0ylmethyl morphine conjugate to lysozyme 47 1.11 O -imidoylmethyl morphine conjugate to glucose (i-phosphate dehydr g e 47 2. Methadone: 2.1 6-keto-7,7,-diphenyl-B-dimethylaminodecanoie acid conjugate to lysozyme 48 3. Meperidine: 3.1 4-carbeth0 -1-carboxymethyl-4-phenylpiperidine conjugate to lysozyme 48 4. Amphetamine: 4.1 N-carboxymethyl amphetamine con ugate to lysozyrne- 49 5. Barbiturates:

5.1 N -carboxyrnethyl phenobarbital conjugate to lysozyme- 50 5.2 -(7-C10t011i0 acid)-5-(2-penty1)-barbituric acid conjugate to lysozy-me 51 5.3 N-canboxymethyl glutethimide 52 5.4 N-( i-carboxybutyl) phenobarbital conjugate to lysozyme 52 5.5 5-(vcrotonic acid)-5-(2-pentyl) barbiturie acid conjugate to lysozyme 53 6. Cocaine:

6.1 Ecgonine conjugate to lysozyme 54 6.2 p-Diazobenzoyl ecgonine con ugate to lysozyme 54 7. Insulin: 7.1 p-Diazobenzamide modified insulin conjug malate dehyd mgenqse 55 8. bteroids:

8.1 Testosterone-3-carboxymethyloxime conjugate to malate dehydr g e 55 8.2 3-(O-carboxymethyl) estradiol conjugate to malate dehydr n 56 EXAMPLE A Preparation of morphine antibodies and binding to support 1. Morphine (900 mg.) was dried for 4 hours at 50 C., 0.01 mm. Hg. The dried morphine was dissolved in 18 ml. of abs. ethanol and 125 mg. sodium hydroxide was added, followed by the addition of 350 mg. dry sodium chlorostirred and refluxed for four hours. The hot solution was treated with 3.8 ml. ethanolic hydrogen chloride (0.85 M) and then filtered while still warm. On cooling overnight, a precipitate (272 mg.) formed which was collected and recrystallized from ethanol/water. On addition of ether to the original filtrate an additional precipitate was obtained which was also recrystallized from ethanol/water. Total yield 600 mg. (55%). On heating this product to 75 C. in vacuo there was a weight loss corresponding to 0.48 molecule of ethanol or 1.15 molecule of water. The dried compound decomposes at 190-220 (depends on rate of heating).

Anal: S H NO Percent theor.: C, 66.45; H, 6.16;

N, 4.08. Percent found: C, 65.87; H, 6.98; N, 4.09, 4.07.

NMR(C5D5N) 2.44 p.p.m. (CH3), 5.08 p.p.m.

2. Carboxymethyl morphine .(240 mg.) suspended in 8 ml. dry dimethyl foramide (DMF) was cooled to -15? ,C. and treated with 84 l isobutyl chloroformate.

The solid dissolved while stirring for 30 minutes at is about 4.0 ml. per rabbit.

ate was added to this solution and the mixture was kept at 0 C. overnight. It was then dialyzed againstdistilled water with four changesv of Water (dialysis 1:.80 lyophilized to give 350 mg. of conjugate.

Hapten concentration on the protein: 7

MWCMM=327 B =41600 M MWBsA=64,4OO js,e. M= 107 o.

and

The ultraviolet spectrum was measured at 280 nm. in

a 1 cm. cell; d=0.59 when the concentration was 0.287

mined from the above data and the formula: a

=C M BsA) XMWc1 1 MWBSA where X number of haptens per molecule, W weight of protein conjugate per liter and MW is the molecular g./l. in water. The degree 'of conjugation can be deter weight where CMM refers to the hapten carboxymethylmorphine, and BSA refers to the protein.

X 46.6 haptens/molecule 3. Antisera may be obtained as follows: The antigen (hapten coupled to an appropriate protein, see above example) is made up in a saline solution (9 g./liter) at a 2 mg./ml. concentration. Per 1.0 ml. aliquot of the above solution, introduced, there is introduced simultaneously 2 ml. of Complete Freunds Adjuvant in homogenized form by means of a two-way needle. For subcutaneous injections, approximately 0.3 ml. (antigen+Freuds solution) is injected per site and for intraperitoneal injections, approximately 0.4 ml. is

tion is added to the serum dropwise with stirring at 4 C.

After standing for 1 hour at that temperature, the solution is centrifuged at 10,000 r.p.m. for 15 minutes and the supernatant removed. The residue is suspended in as small a volume as possible of 1x BB (borate buffer, see below for description), transferred to a dialysis bag and dialyzed overnight against BB, pH 8.0 The residue in the dialyzed bag is then isolated and frozen.

(To made borate buffer, dissolve 24.6 g. boric acid in water, adjust the pH with sodium hydroxide to a pH 7.98.0, add 0.1 g. of sodium azide and 0.01 g. of ethylmercurithiosalicylate and bring the'total volume to o'ne '2 liter.

4. Into 20 ml. of dimethyl formamide is introduced 400 mg. aminoethyl-Bio-Gel P-300 and 300 mg. of carboxymethyl morphine (see Example A-1) and 1 g. 'sodium bicarbonate is added. After stirring the suspension for two days at 4 C., the suspension is filtered, the residue is washed with water until the washings are neutral, and then the residue is dried in vacuuml i I The resulting product is then suspended-in. 20 ml. rabbit serum containing morphine antibodies and is stirred for 4 hours at 4 C. Filtration gives a residue which is resuspended in -5 ml. 'phthalate butter, pH 3.8..:(0.1 M)

of substantially pure antibodies.

5. Into a flask were cornbined 5 m1. Sepharose ZBSUS-Q pension, 5 ml. water, and 5 ml. mg./ml. vCNBr 80111-9 tion and the pH adjusted to 11.5 with 4N NaOH. While stirring the mixture, the pH was metered and maintained at 11-11.5 with 4 N NaOH until no further change in pH.

injected. The total dosage 

